Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene
DNA repair protein -methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the -position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is...
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Published in | Journal of biological methods Vol. 5; no. 2; p. e92 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Journal of Biological Methods
07.06.2018
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Subjects | |
Online Access | Get full text |
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Summary: | DNA repair protein
-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the
-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression. These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing, validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments. Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing interests: The authors have declared that no competing interests exist. Current address: Department of Microbiology, Govt. Medical College, Kannauj, Uttar Pradesh, India Abbreviations used: AFLPs, amplified fragment length polymorphisms; AS, allele-specific; Bu, busulfan; DNA, deoxyribonucleic acid; dNTP’s, deoxynucleotide triphosphates; EDTA, ethylenediaminetetraacetic acid; HPLC, high performance liquid chromatography; HRM, high resolution melting; HSCT, hematopoietic stem cell transplantation; MGMT, methylguanine-DNA methyltransferase; PCR, polymerase chain reaction; RAPDs, randomly amplified polymorphic DNAs; RFLPs, restriction fragment length polymorphisms; SNPs, single nucleotide polymorphisms; WASP, web-based allele-specific primer |
ISSN: | 2326-9901 2326-9901 |
DOI: | 10.14440/jbm.2018.224 |